Not applicable.
1. Field of the Invention
The present invention relates generally to methods and devices for determining properties of a medium using radiation sensors that are compensated for component drift and changes in radiation coupling to the medium. A specific embodiment of the invention relates to biomass monitoring, where radiation scattering is used to measure the concentration of cells or microorganisms in liquid cultures.
2. Description of Prior Art
Radiation sensors are widely used in the analysis of material properties. In particular, the absorption and scattering of radiation, measured by means of a radiation sensor, can be related to the concentration of a particular material within a mixture. Such measurements can be made rapidly and free from the risk of consuming, infecting, or damaging the material being analyzed. Some examples of commercial applications of optical radiation sensors are: smoke detectors, stack emission sensors, tissue oxygenation sensors, and biomass monitors in liquid cultures of cells or microorganisms.
Two common difficulties with radiation sensors that have been addressed in prior art, but not adequately resolved in combination, are sensor drift and limited dynamic range. Drift of sensor response can result from of any of the following: (1) a change in source intensity, (2) a change in detector sensitivity, and (3) a change in the efficiency of radiation coupling between the sensor and the sample. As radiation sources and detectors age, their respective output and sensitivity inevitably change. These aging effects can lead to inaccuracy in the determination of the property of the material being analyzed. The third source of sensor drift can be particularly prevalent in sensors that monitor harsh, dirty, or biologically active environments such as in the above examples of stack emission monitoring and biomass monitoring of liquid cultures. In both of these examples, accumulation of matter on the surface of the sensor can lead to drift or inaccurate readings of the sensor.
The dynamic range of material properties that can be measured by many radiation sensors is limited by the non-linear relationship between concentration of the material and attenuation of the radiation. For differing concentrations of an absorbing or scattering material of interest, the radiation level impinging upon the sensor will vary widely; high concentrations of material will greatly attenuate the radiation compared to low concentrations. For this reason, sensors that measure the transmission of radiation through a single fixed path length of material will have an inherently narrow dynamic range over which material properties can be measured.
U.S. Pat. No. 3,976,891xe2x80x94Parkinson discloses a sensor for smoke detection that compensates for changes in the optical coupling efficiency between the sensor and medium of interest by comparing the light transmitted through two different path lengths of air to two different detectors. The disadvantage of this method is that it provides no compensation for light source or detector drift. Further, the dynamic range of the measurement is limited by its reliance on only light transmittance to determine smoke density.
U.S. Pat. No. 4,981,362xe2x80x94deJong discloses a particle concentration measuring method employing a single light source and detector. Light is transmitted from the source to the detector through a movable window. By computing the ratio of the light transmitted through the window at two different path length settings, light source, detector, and optical coupling drift are partially compensated. However, any instrumental drift that occurs between the two measurements will not be compensated. Another disadvantage of this method is that it requires the use of a moving part whose mechanical motion and physical manufacture must be highly reproducible in order to compare measurements made at different times or with different devices. This method also suffers from limited dynamic range due to reliance on only light transmittance to determine particle concentration.
U.S. Pat. No. 5,617,212xe2x80x94Stuart describes an apparatus for open-path gas monitoring that measures transmission of light between two sources and two detectors. The second source is for calibration purposes only and does not pass through the sample. Likewise, the second detector is used only for calibration and measures light from the two sources that does not pass through the samples. This method provides the advantage of compensating for light source and detector drift. However, it does not provide compensation for changes in optical coupling efficiency between the sensor and sample. In addition, this method suffers from both limited sensitivity and dynamic range due its use of only light transmittance to measure the gas density.
U.S. Pat. No. 5,497,769xe2x80x94Gratton describes a sensor employing multiple light sources and a single detector. U.S. Pat. No. 5,529,065xe2x80x94Tsuchiya describes a sensor employing a single light source and multiple detectors. Both patents describe the measurement of light diffusely reflected from highly scattering materials. The disadvantage of these methods is that they require frequent re-calibration to compensate for light source and detector drift. In addition these methods are prone to error due to changes in the optical coupling efficiency between the sensor and sample.
U.S. Pat. Nos. 4,017,193xe2x80x94Loiterman and U.S. Pat. No. 5,482,842xe2x80x94Berndt, and European Patent Application 0945100A1xe2x80x94Hueber describe sensors employing two light sources and two detectors arranged to provide a pair of equal-length long paths and a pair of equal-length short paths between light sources and detectors. The four signals provided by these four combinations of sources and detectors are combined in a manner that compensates for drift in light source intensity, detector sensitivity, and efficiency of optical coupling between the sensor and the sample. The methods described by Loiterman and Berndt involve transmitting light through gaseous materials and detecting the extent of scattering or absorption, respectively. The method described by Hueber involves the detection of diffusely reflected light from a highly scattering medium such as tissue. The critical disadvantage of all three of these methods is the limited dynamic range over which material properties can be measured due to the geometric constraint that there be only two unique path lengths between light sources and detectors.
Liquid cultures of cells or microorganisms are frequently grown for research purposes or for commercial gain. Cells or microorganisms can be genetically modified to produce high yields of chemicals that may be difficult, expensive, or impossible to synthesize by other means. In order to prevent growth of other undesirable cells or microorganisms in the same liquid culture, it is important that the culture be grown under sterile conditions. For this reason, the growth medium is sterilized prior to inoculation with the desired cell or microorganism. In order to maintain a barrier to foreign organisms and optimize the growth of the desired cell or microorganism, liquid cultures are frequently grown under highly controlled conditions in what are referred to as fermenters or bioreactors. In addition to maintaining sterile conditions, fermenters may provide control over such parameters as temperature, pH, rate of stirring, and concentration of nutrients and dissolved gases.
Cells or microorganisms typically undergo several stages of growth in a fermenter. After inoculation, the initial growth rate of the cells or microorganisms may be slow, as the organism becomes accustomed to the new environment. This is frequently followed by a rapid growth phase where the biomass increases nearly exponentially. This growth period is sometimes referred to as the xe2x80x9clog phasexe2x80x9d due to the fact that the change in the logarithm of biomass is nearly linear with time. Eventually, as the nutrient supply relative to the biomass diminishes, the growth will slow. In order to achieve maximum biomass, the conditions in the fermenter need to be changed during the different phases of growth. Ideally a feedback mechanism would link the measured growth of the cells or microorganisms to the conditions in the fermenter. Frequently, a physical or chemical stimulus is used to induce production of a desired chemical by the cells or microorganisms. The timing of this induction relative to the growth cycle of the cells or microorganisms is often critical in order to achieve maximum chemical yield. Unfortunately, methods of continuously and reliably measuring the growth of cells or microorganisms in liquid cultures are not widely available.
The most commonly used method of measuring the biomass in liquid cultures is by extracting a portion of the liquid and measuring its optical density with a spectrophotometer. This method has several disadvantages: (1) each time liquid is withdrawn, there is a risk that the culture will be contaminated, (2) the method is not continuous, and (3) the method is labor intensive, requiring frequent extraction and precise volumetric dilution of the extracted liquid when high cell concentrations are measured. Commercial devices are available (eg. Wedgewood Technology, Incorporated, Model 650 xe2x80x9cAbsorbance Monitorxe2x80x9d) that offer continuous measurement of optical density using a probe that is immersed in the liquid culture. Unfortunately, such devices are prone to drift (see above discussion of compensated radiation sensors), particularly due to growth of cells or microorganisms on the sensor itself In addition, the range of biomass that can be measured is severely limited by the use of fixed path length transmission measurements. Many microorganisms, particularly strains of yeast (e.g. Pichia Pastoris), are grown to much higher concentrations (e.g. 50 g/L and higher) than can be measured with any known commercially-available device based on optical transmittance.
U.S. Pat. No. 5,483,080 xe2x80x94Tam describes a method for measuring biomass in liquid cultures using optical reflectance. The method restricts the source or detector to be singular in number. By measuring the light that is diffusely reflected back from the liquid culture, the method gains an advantage in the range of cell densities that can be measured, compared to transmission methods. However, the sensor output is highly non-linear with change in concentration or the logarithm of concentration of cells or microorganisms. In one embodiment the effect of detector drift is compensated by calculating a ratio of the light detected from two light sources. However, this method does not compensate for light source drift or for changes in efficiency of optical coupling between the sensor and sample. One embodiment of this invention is a sensor designed to be immersed in a liquid culture. Growth of cells or microorganisms on the surface of the sensor may lead to changes in sensor response that do not accurately reflect the suspended cell concentration. No means is provided to compensate for this effect. A second embodiment of this invention is a sensor mounted to the exterior of a container holding a liquid culture. The container is assumed to have a window that is transparent to the light source. This non-contact method provides the advantage that the sensor does not need to be sterilized. However, no means is provided to compensate for optical imperfections in the container wall, either inherent to the window or as caused by the growth of cells or microorganisms on the interior of the window over time. In addition, the invention provides no method of compensating for the variable glass thickness that is observed between different types of growth vessels. A specific calibration would need to be provided for each type of vessel on which the method is practiced.
The present invention provides a radiation sensing method and device that can be used to measure physical properties of materials over a wide dynamic range. The measurements are self-compensated for both component drift and changes in radiation coupling efficiency between the sensors and the material of interest. The invention finds particular utility in measuring biomass in liquid cell cultures with high accuracy over a wide dynamic range. The measurement can be made with the sensor external to the liquid culture container in a manner that is compensated for the thickness of the container window.
Accordingly, several objects and advantages of the present invention are:
a) the measured values of the physical properties being determined are compensated for fluctuations or the effects of aging on the source radiation intensity,
b) the measured values of the physical properties being determined are compensated for fluctuations or the effects of aging on the detector sensitivity,
c) the measured values of the physical properties being determined are compensated for dirt or other materials or conditions that effect the interface between the sensor and the material of interest,
d) the necessity of using moving parts to confer advantages a) through c) is eliminated, and
e) the physical properties are measurable over a wider dynamic range compared to other radiation sensing methods that confer advantages a) through d).
Additional objects and advantages of the present invention for the specific embodiment in which the radiation sensor is used to measure particle concentration in scattering media:
a) the radiation sensor output provides a more linear response to changes in the logarithm of concentration than do radiation sensors described in prior art, and
b) the sensor output is compensated for the accumulation of particles at the interface between the sensor and the scattering medium, and
c) the concentration of multiple particle types and/or the particle size distribution of particular particle types may be simultaneously measured.
Additional objects and advantages of the present invention for the specific embodiment in which the sensor is mounted externally to a container holding a liquid culture for the purpose of measuring the biomass:
a) the need to sterilize the sensor and its housing are eliminated,
b) the sensor housing can be constructed for lower cost than sensor housings requiring sterilization and immersion in a liquid culture,
c) the risk of contaminating the liquid culture with foreign matter is eliminated,
d) multiple different fermenters can be monitored with the same sensor without interrupting the growth of the cultures or risking exposure of the cultures to foreign matter, and
e) the measured value of the biomass in the liquid culture is compensated for variations in window thickness between different fermenters.